Pre-equilibration kinetic size-exclusion chromatography with mass spectrometry detection (peKSEC-MS) for label-free solution-based kinetic analysis of protein–small molecule interactions

The Analyst ◽  
2015 ◽  
Vol 140 (4) ◽  
pp. 990-994 ◽  
Author(s):  
Jiayin Bao ◽  
Svetlana M. Krylova ◽  
Leonid T. Cherney ◽  
J. C. Yves Le Blanc ◽  
Patrick Pribil ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Kinetic Analysis ◽  
Small Molecule ◽  
Size Exclusion ◽  
Free Solution ◽  
Label Free ◽  
Mass Spectrometry Detection ◽  
Reversible Binding ◽  
Exclusion Chromatography ◽  
Molecule Interactions

Label-free solution-based kinetic analysis of reversible binding between protein and small molecule.

scholarly journals Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 246 ◽  
Author(s):  
Anders Askeland ◽  
Anne Borup ◽  
Ole Østergaard ◽  
Jesper V. Olsen ◽  
Sigrid M. Lund ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
High Speed ◽  
Proteome Analysis ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Label Free ◽  
Protein Biomarkers ◽  
Transmission Electron ◽  
Exclusion Chromatography

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

scholarly journals A Chemoinformatics Analysis of Hit Lists Obtained from High-Throughput Affinity-Selection Screening

2005 ◽  
Vol 11 (2) ◽  
pp. 123-130 ◽  
Author(s):  
Nathan Brown ◽  
Hartmut Zehender ◽  
Kamal Azzaoui ◽  
Ansgar Schuffenhauer ◽  
Lorenz M. Mayr ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Size Exclusion ◽  
Label Free ◽  
Affinity Selection ◽  
High Performing ◽  
Exclusion Chromatography ◽  
Screening Platform ◽  
Screening Technology

The high-throughput affinity-selection screening platform SpeedScreen was recently reported by the Novartis Institutes for BioMedical Research as a homogeneous, label-free screening technology with mass-spectrometry readout. SpeedScreen relies on the screening of compound mixtures with various target proteins and uses fast size-exclusion chromatography to separate target-bound from unbound substances. After disintegration of the target-binder complex, the binder molecules are identified by their molecular masses using liquid chromatography/mass spectrometry. The authors report an analysis of the molecular properties of hits obtained with SpeedScreen on 26 targets screened within the past few years at Novartis using this technology. Affinity-based SpeedScreen is a robust high-throughput screening technology that does not accumulate frequent hitters or potential covalent binders. The hits are representative of the most commonly identified scaffold classes observed for known drugs. Validated SpeedScreen hits tend to be enriched on more lipophilic and larger-molecular-weight compounds compared to the whole library. The potential for a reduced SpeedScreen screening set to be used in case only limited protein quantities are available is evaluated. Such a reduced compound set should also maximize the coverage of the high-performing regions of the chemical property and class spaces; chemoinformatics methods including genetic algorithms and divisive K-means clustering are used for this aim.

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